Structure And Mechanism Of Human DNA Polymerase η

Life on earth can scarcely escape the ultraviolet radiation (UV) of sunlight, which catalyzes covalent linkages between adjacent pyrimidines in DNA. The resulting pyrimidine dimers are roadblocks to most DNA polymerases, but the human POLH gene, defective in the variant form of xeroderma pigmentosum (XPV), encodes a Y-family DNA polymerase, Pol η, specialized for translesion synthesis (TLS) through cyclobutane pyrimidine dimers (CPDs). XPV is characterized by sunlight-induced pigmentation changes and a highly elevated incidence of skin malignancies. Moreover, Pol η is the only one of the fifteen human DNA polymerases in which defects are unequivocally associated with cancer. Among human Y-family polymerases (Revl, Pols η, ι and κ), the lesion-bypass specificity and efficiency of Pol η stand out. Pol η binds CPD-containing DNA better than undamaged DNA and extends primers more processively opposite and up to2nt beyond CPDs.Structurally, the major species of cisplatin-crosslinked guanines (Pt-GG) is related to CPD. Among many polymerases tested in vitro, human Pol η is the most efficient and accurate at bypassing Pt-GG lesions. Also, it has been shown that human Pol η participates somatic hypermutation in B cells, which introduces very high mutation rate at WA motif. To elucidate the mechanisms, we co-crystallized human Pol η with substrate DNA and incoming nucleotide. Combined with biochemistry assay and kinetics measurement, we answer these questions and proposed a new model for new anti-cancer drug desigsn as well as its D-loop bypassing activity. The main conclusions are:1. We were able to express, purify and co-crystallize human Pol η in ternary form.2. The crystals were diffracted to beyond1.7A and structure was determined using molecular replacement and MAD.3. Human Pol η acts like a "molecular splint" to stabilize damaged DNA in a normal B-form conformation. An enlarged active site accommodates the thymine dimer with excellent stereochemistry for two-metal ion catalysis. Two residues conserved among Pol η orthologs form specific hydrogen bonds with the lesion and the incoming nucleotide to assist translesion synthesis. Based on the structures, eight human Pol η missense mutations causing XPV can be rationalized as undermining the "molecular splint" or perturbing the active-site alignment.4. Structural and biochemistry analysis show why human Pol η is inefficient at extending primers after cisplatin lesions, which necessitates a second translesion DNA polymerase to complete bypass in vivo. A hydrophobic pocket near the primer-binding site in human Pol η is identified as a potential drug target for inhibiting translesion synthesis and thereby reducing chemoresistance.5. For somatic hypermutation, WA motif is the "hot spot" and human Pol η do prefer to insert dGTP which correlate with in vivo assay. A hydrogen bond network was observed to stabilize the T-G mismatch in the active site.